Interpretive Summary: Worms that infect livestock cause disease and economic losses estimated at over 500 million dollars annually. The effectiveness of drugs that are currently available for control of these diseases is limited because of cost and because drug resistance can occur. The lack of knowledge about basic biology of parasitic worms has hampered development of new control strategies. The current paper studies the characteristics of excretory/secretory products released by the sheep stomach worm, Haemonchus contortus. These studies reveal that fourth stage larvae and adult worms release specific proteolytic enzymes that may help the worm adapt to the host. Studies suggest that this enzyme may inhibit blood coagulation. Since the stomach worm is a blood feeding parasite, inhibition of this enzyme may provide the basis for development of new controls.

Technical Abstract:
Cysteine protease activity was present in media collected after 24-hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC) substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with Mr values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4 but not L3 larval stage. The active and developmentally-regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition and immune evasion.